Specific Association of Cyclin-like Uracil-DNA Glycosylase with the Proliferating Cell Nuclear Antigen

Abstract

We have previously isolated a human gene that encodes a cyclin-like protein with uracil-removing activity (UDG2) (Muller, S. J., and Caradonna, S. 1993.J. Biol. Chem.268, 1310–1319). The structural and regulatory similarities shared between this uracil-DNA glycosylase and cyclins suggested that it may interact with additional proteins. Using a unique affinity purification protocol (Ugi-Sepharose) and anti-UDG2 antibodies, we have identified a physical interaction between the cyclin-like uracil-DNA glycosylase and PCNA in extracts derived from HeLa cells. Conversely, we show that anti-PCNA immunoprecipitates possess significant uracil-DNA glycosylase activity. This activity is specifically blocked by the addition of uracil-DNA glycosylase inhibitor protein (Ugi) derived from bacteriophage PBS2. To further characterize this association, we performedin vitromixing experiments using35S-labeled PCNA and uracil-DNA glycosylase (UDG2) that were generated in a coupled transcription/translation system. We show that UDG2 and PCNA are coprecipitated using anti-PCNA antibodies and anti-UDG2 antibodies as well as Ugi-Sepharose. When PCNA is preincubated with synthetic peptides corresponding to amino acid residues 73–90 of UDG2, the PCNA-UDG2 association is prevented. By contrast, addition of synthetic peptides corresponding to amino acid residues 208–223 has no effect on this interaction. These findings suggest that the UDG2 domain encompassing amino acids 73–90 is directly involved in binding PCNA.